Pre-mRNA splicing is one of the most important points of regulation of eukaryotic gene expression. Although the overall chemistry of the reaction is likely to be highly conserved in eukaryotes, the catalysts of the process, namely proteins and small nuclear RNAs (snRNAs), have primary sequences that are only partially conserved evolutionarily. It is proposed herein to initiate a study of the snRNAs of a primitive protist (the enteric ameba Entamoeba histolytica) and primitive animal (the cestode flatworm Taenia solium). The specific aim of this work is to uncover the differences in snRNA structure and function between these species and the more commonly studied human and yeast splicing systems. The overall objectives of this work are to increase our understanding of the molecular biology of E. Histolytica and T. Solium, which are human parasites compromising the health of millions of people worldwide. By this effort, it is hoped that points of evolutionary divergence in pre-mRNA splicing can be found, between parasite and host, that may ultimately be exploited for rational drug design. Students will characterize and isolate the snRNAs of E. histolytica and T. solium, by polyacrylamide gel electrophoresis, and prepare cDNA copies using oligonucleotide primers to conserved regions. They will use these data to build secondary structure models for the snRNAs, and test their models statistically by searching for "compensatory mutations" in snRNA genes of phylogenetically related species, such as E. disbar and T. saginata. These experiments will allow the students to learn basic principles of evolution, RNA thermodynamics, and molecular cloning. To study the relationship between snRNA structure and function, some snRNA domains may be tested functionally (in future work, not included in this proposal) by transfection into Saccharomyces cerevisiae. The mentoring of minority students is a major goal of this proposal, and seven consultants and collaborators have offered to engage in ongoing discussions with the students about their research and career plans. These consultants/collaborators and outstanding scientists/physicians, and are leaders in the fields of Entamoeba and Taenia research and clinical medicine, and RNA splicing/snRNA phylogeny. Although these scientists will not be directly involved in the research project, they will help to foster the intellectual development of the students and encourage their maturation as scientists.